Polysaccharides containing uronic acid residues are found in virtually every species in Nature. These polymers include plant polysaccharides such as pectin, alginic acid, and many gums and mucilages; animal polysaccharides such as the glycosaminoglycans; and bacterial polysaccharides such as a variety of capsular materials. In such polysaccharides, the uronic acid residues may account for 10 to 100 percent of the total monosaccharide residues in the polymer. Uronic acid types that are found in these polymers include D-glucuronic, D-galacturonic, D-mannuronic, L-iduronic, and L-guluronic acids.
A number of animal uronic acid-containing polysaccharides such as heparins, chondroitin sulfates, and dermatan sulfates occur naturally with sulfate substituents in various positions on both their uronic acid and their hexosamine residues. Interestingly, the presence of sulfate groups on these uronic acid-containing polymers leads to species that have various biological and therapeutic activities. For example, heparin exhibits important anticoagulant and antithrombotic activities, whereas the bacterial E. coli K5 polysaccharide, which has a structure like that of heparin but which is not sulfated, lacks these activities. Similarly, chondroitin sulfate and dermatan sulfates have activities similar to those of heparin, whereas their structurally-analogous E. coli K4 polysaccharide, which is not sulfated, lacks these activities. In the heparin case, it has been demonstrated that both the uronic acid carboxyl groups and the sulfates are essential for the anticoagulant activity. Consequently, sulfation of unsulfated uronic acid-containing polysaccharides may convert such polymers to biologically active species. Similarly, it may be possible to generate substances with unique activities by further sulfating polymers that occur naturally as partially sulfated polysaccharides.
Two general approaches have been used extensively to sulfate polysaccharides. These include (a) the use of amine conjugates of sulfur trioxide as the sulfating agent (Gilbert (1962) Chem. Rev. 62: 550-589; Nagasawa, et al. (1986)Carbohyd. Res. 158: 183-190; Casu, et al. (1994) Carbohyd. Res. 263: 271-284), and (b) the use of a mixture of sulfuric acid and chlorosulfonic acid as the sulfating agent (Naggi, et al. (1987) Biochem. Pharmacol. 36: 1895-1900). We report here a new method for sulfation of uronic acid-containing polysaccharides. This method involves the generation of a sulfating reagent by activation of sulfate ions with carbodiimides. Such active sulfates react with the hydroxyl groups of polysaccharides, generating products with degrees of sulfation that can be controlled by modulation of the reaction conditions. The sulfuric acid/carbodiimide method described here offers a number of advantages over sulfation methods that have been reported in the scientific and the patent literature. As one example the sulfuric acid/chlorosulfonic acid reagent causes depolymerization of the polysaccharide as the sulfation proceeds so that the molecular weight of the final product is difficult to control; the carbodiimide/sulfuric acid method does not cause any depolymerization.
Previous reports have described the use of sulfuric acid and a carbodiimide to sulfate monosaccharides (Mumma, et al (1970) Carbohyd. Res. 14: 119-122; Takano, R., T. Ueda, et aL (1992) Biosci. Biotech. Biochem. 56: 1413-1416) and neutral polysaccharides (Takano, R., S. Yoshikawa, et al. (1996) J. Carbohyd. Chem. 15: 449-457). In these reports it was shown that C6 of the monosaccharides was sulfated much more rapidly than the secondary hydroxyls, but a high level of sulfation of these carbohydrates, i.e.  greater than 1 sulfate per monosaccharide residue, could not be achieved. Furthermore, this method was not applied to uronic acid-containing polysaccharides, where the use of the carbodiimide-activated sulfate reagent presents some special problems. First of all, carbodiimides activate the carboxyl groups of the uronic acid-containing polysaccharides, rendering them reactive with a variety of nucleophiles that might be present in the reaction mixture (amines, alcohols, or hydrides (Hoiberg and Mumma (1969) J. Am. Chem. Soc. 91: 4273-4278; Danishefsky, and Siskovic (1971) Carbohyd. Res. 16: 199-205; Taylor and Conrad (1972) Biochemistry 11: 1383-1388), yielding undesired products. Thus, for example, carbodiimide-activated uronic acid residues may react with hydroxyl groups on adjacent chains of the polysaccharide in the reaction mixture to form ester cross-links, thus increasing the molecular weight of the products. Also, the sulfate groups that are already present on the substrates to be sulfated may be activated by the carbodiimide, rendering them quite strong electrophiles and allowing sulfate cross-linking of the polymer chains. Furthermore, during the sulfation reaction, the formation of mixed anhydrides of the sulfuric acid and carboxylic acids may occur. In addition to yielding undesired products, these side reactions consume the sulfation reagent, lowering the concentration of the reagent and thus the extent of sulfation of the desired product. Thus, for the sulfation reaction, it is important to prevent side reactions so that the polymer is altered only by the addition of sulfates to the alcoholic hydroxyl groups. The method described here uses conditions that have been developed to minimize the side reactions and to control the degree of sulfation, yielding, maximally, greater that two sulfate residues per monosaccharide.
The present invention provides news methods for the preparation of sulfated (or oversulfated or hypersulfated) uronic acid-containing polysaccharides, including glycosaminoglycans. In such methods, tertiary or quaternary alkyl amine salts of uronic acid-containing polysaccharides are dissolved in an aprotic solvent, e.g. dimethylformamide (DMF), and an N,N-carbodiimide and sulfuric acid are added. The sulfuric acid is activated by the carbodiimide and the free hydroxyl groups of the heparinoid react readily with these activated anions. The methods described herein work efficiently for uronic acid-containing polysaccharides, and are superior to previously used carbodiimide/sulfuric acid methods described for monosaccharides and neutral polysaccharides, wherein the extent of sulfation was limited to  less than 1 sulfate/monosaccharide. With the previously reported, more limited reaction conditions, the sulfation occurred primarily at C6 positions of the monosaccharide residues, i.e., positions that are already extensively sulfated in many of the naturally-occurring sulfated polysaccharides, including chondroitin 6-sulfate and heparinoids. The present invention provides methods whereby the conditions employed minimize the modifications of the carboxyl groups while maximizing the degree of sulfation. In the methods of the present invention, the carbodiimide or sulfate derivatives of the carboxylic acids formed during the reaction are converted back to the original carboxylic acids. Furthermore, the polysaccharides do not become degraded or cross-linked during the reaction.
More particularly, the present invention provides a process for the preparation of sulfated uronic acid-containing polysaccharides, the process comprising: (1) converting the uronic acid-containing polysaccharide into an amine salt, and (2) 0-sulfating the amine salt of the uronic acid-containing polysaccharide with carbodiimide and sulfuric acid. The first step, wherein the alkali metal salt of the uronic acid-containing polysaccharide is converted to an amine salt, can be done by either ion exchange chromatography or by the addition of a solution of the tertiary or quaternary amine to an about 2% to about 10% solution of the uronic acid-containing polysaccharide.
In one embodiment, the present invention relates to methods for the preparation of oversulfated heparinoids, the methods generally comprising: (1) conversion of the heparinoids into solvent-soluble salts; (2) 0-sulfation of the heparinoids with carbodiimide and sulfuric acid; and (3) N-sulfation of 0-sulfated heparinoid. Quite surprisingly, the resulting heparinoids have a high degree of sulfation, up to 4 sulfates per disaccharide.
The use of certain terms in this specification preferably includes reference to the products or techniques defined below in relation to those terms. xe2x80x9cMonosaccharide,xe2x80x9d as used herein, refers to a polyhydroxy alcohol containing either an aldehyde or a ketone group, i.e., a simple sugar. Monosaccharide includes reference to naturally occurring simple sugars as well as simple sugars which have been chemically modified. Modified monosaccharides include, but are not limited to, monosaccharides that have increased or decreased sulfation or that have modified carboxyl, amino or hydroxyl groups.
xe2x80x9cUronic Acid,xe2x80x9d as used herein, refers to a monosaccharide in which the primary alcoholic carbon is replaced by a carboxyl group.
xe2x80x9cPolysaccharide,xe2x80x9d as used herein, refers to a linear or branched polymer of more than 10 monosaccharides that are linked by means of glycosidic linkages.
xe2x80x9cOligosaccharide units,xe2x80x9d as used herein, refers to a linear or branched polymer of more than 2 monosaccharides that are linked together by means of glycosidic linkages.
xe2x80x9cPolyanion,xe2x80x9d as used herein, refers to a molecule that possesses a large number of negative charges. xe2x80x9cPolyanionic carbohydrates,xe2x80x9d as used herein, includes reference to carbohydrates that possess a large number of negative charges.
xe2x80x9cGlycosaminoglycan,xe2x80x9d as used herein, includes reference to a polysaccharide composed of repeating disaccharide units. The disaccharides always contain an amino sugar (i.e., glucosamine or galactosamine) and one other monosaccharide, which may be a uronic acid (i.e., glucuronic acid or iduronic acid) as in hyaluronic acid, heparin, heparan sulfate, chondroitin sulfate or dermatan sulfatexe2x80x94or galactose as in keratan sulfate. The glycosaminoglycan chain may be sulfated on either moiety of the repeating disaccharide.
xe2x80x9cHeparinoids,xe2x80x9d as used herein, refer to all structural variations of heparin and heparan sulfate. These are oligomers of at least one disaccharide or polymers of at least 18 different disaccharides, which represent their monomeric units. The disaccharides are made up of a hexuronic acid residue and a D-glucosamine residue which are linked to each other and to the other disaccharides by 1xe2x86x924 linkages. The glucoasamine unit may be either N-acetylated (GlcNAc) or N-sulfated (GlcNSO3). The uronic acid may be either a xcex2-D-glucuronic acid or an xcex1-L-iduronic acid residue. O-Sulfate substituents are found at C2 of some of the uronic acid residues and at C6 of some of the glucosamine residues. A general feature of these structures is that blocks of uronic acidxe2x86x92glucosamine disaccharides that contain high degrees of sulfation are separated from other such blocks by blocks of unsulfated glucosamine xe2x86x92N-acetylated glucosamine disaccharides. The relative length of the sulfated and unsulfated blocks differ for different heparinoids, with relatively few unsulfated disaccharides in heparins and many blocks of GlcAxe2x86x92GlcNAc disaccharides in heparan sulfates. Thus, in all heparinoids there are a number of unsubstituted hydroxyl groups on both the glucosamine and the uronic acid residues.
xe2x80x9cHeparinxe2x80x9d (or, interchangeably, xe2x80x9cstandard heparinxe2x80x9d (SH) or xe2x80x9cunmodified heparin,xe2x80x9d as used herein, includes reference to heparinoids that are highly sulfated and that have relatively high IdoA/GlcA ratios (1 to 10) and GlcNSO3/GlcNAc ratios (1 to 10). Generally, heparin has an average molecular weight ranging from about 6,000 Daltons to 40,000 Daltons with an average of about 12,000 Daltons, depending on the source of the heparin and the methods used to isolate it.
xe2x80x9cHeparan Sulfatexe2x80x9d (HS), as used herein, includes reference to heparinoids that are less highly sulfated and that have relatively low IdoA/GlcA ratios (0.5-1.5) and GlcNSO3/GlcNAc ratios (0.5-1.5).
xe2x80x9cDermatan Sulfatexe2x80x9d (DS), as used herein, includes reference to a heterogeneous glycosaminoglycan mixture that contains disaccharide repeat units consisting of N-acetyl-D-galactosamine and D-glucuronic acids, as well as disaccharide repeat units consisting of N-acetyl-D-galactosamine and L-iduronic acid. The N-acetyl-D-galactosamine residues may be sulfated on the 4 and/or the 6 position. The uronic acids are present with variable degrees of sulfation.
xe2x80x9cChondroitin sulfatexe2x80x9d (CS), as used herein, includes reference to a heterogeneous glycosaminoglycan mixture that contains disaccharide repeat units consisting of N-acetyl-D-galactosamine and D-glucuronic acids. The N-acetyl-D-galactosamine residues may be sulfated on the 4 and/or the 6 position.
xe2x80x9cHyaluronic acidxe2x80x9d, as used herein, includes reference to a heterogeneous glycosaminoglycan mixture that contains disaccharide repeat units consisting of N-acetyl-D-glucosamine and D-glucuronic acids.
xe2x80x9cAlginic acidxe2x80x9d, as used herein, includes reference to a heterogeneous polysaccharide mixture that contains L-glucuronic acids and manuronic acids.
The present invention provides processes for the preparation of sulfated uronic acid-containing polysaccharides, including oversulfated glycosaminoglycans, the methods generally comprising conversion of the polysaccharides into aprotic solvent soluble salts, O-sulfation of the polysaccharides with carbodiimide and sulfuric acid and, in the case of the heparinoids, N-sulfation of the O-sulfated product. The specific conditions employed during the above-described process enable one to obtain a surprisingly high degree of sulfation of the polysaccharides, i.e., up to 4 sulfates per monosaccharide. Earlier methods have described using a carbodiimide and sulfuric acid to sulfate monosaccharides and neutral polysaccharides. Although these methods demonstrated that primary hydroxyl groups were sulfated much more rapidly than the secondary hydroxyls, the conditions developed could not give a high level of sulfation of these carbohydrates, i.e., greater than 1 sulfate per monosaccharide residue. Furthermore, they did not address the special concerns required for sulfation of uronic acid-containing polysaccharides.
In the first step of the process of the present invention, an alkali metal salt, e.g., sodium, salt of a uronic acid-containing polysaccharide is converted to an amine salt, such as tributylamine salt, or to a long chain quatemary amine salt. Examples of amine salts suitable for use in the present invention include, but are not limited to, the following: trimethylamine, triethylamine, tripropylamine, tributylamine and quatemary ammonium salts. In a preferred embodiment, the amine salt is a tertiary amine salt such as tributylamine salt. In another preferred embodiment, the amine salt is a quaternary ammonium salt, such as cetylpyridium and cetyltrimethylammonium salts. These amine salts can be obtained by various ion exchange methods or by precipitation approaches:
The uronic acid-containing polysaccharide can be converted to its amine salt using standard methods and procedures known to and used by those of skill in the art. For instance, the tertiary amine salt can be obtained by ion exchange chromatography or, alternatively, by batch ion-exchange. More particularly, to generate the tertiary amine salt of the uronic acid-containing polysaccharide by ion exchange chromatography, the uronic acid-containing polysaccharide in cooled (2-8xc2x0 C.) double distilled water is loaded onto an ion exchange column at refrigeration temperature (2-8xc2x0 C.). The eluent from the column is then neutralized with a tertiary amine, such as tributylamine (TBA) or tripropylamine (TPA). The mixture is then lyophilized to obtain the tertiary amine salt of the uronic acid-containing polysaccharide. In a preferred embodiment, a Dowex 50W-X8 resin 20-50 mesh, ion exchange column is used. In addition, to generate the tertiary amine salt of the uronic acid-containing polysaccharide by batch ion-exchange, the uronic acid-containing polysaccharide in double distilled water is mixed with roughly the same volume of an ion exchange resin at refrigeration temperature (2-8xc2x0 C.) with constant stirring. The mixture is then filtrated and neutralized by the addition of the tertiary amine, such as tributylamine (TBA) or tripropylamine (TPA). The mixture is then lyophilized to obtain the tertiary amine salt of the uronic acid-containing polysaccharide.
Alternatively, the sodium salt of the uronic acid-containing polysaccharide can be converted to a quaternary amine salt, preferably a long chain quatemary amine salt. In this embodiment, a quaternary amine, salt such as cetylpyridium chloride (CPC) or cetyltrimethylammonium bromide, is dissolved in double distilled water to make about a 5% to about a 15% solution and, more preferably, about a 10% solution. The quatemary amine salt solution is then added to about a 2% to about a 10% solution of the uronic acid-containing polysaccharide in about 5 mM to about 25 mM Na2SO4 until no further precipitation is formed. The precipitate is collected by centrifugation or filtration, and lyophilized to obtain the quaternary amine salt of the uronic acid-containing polysaccharide.
In step two of the above process, a first solution is prepared by contacting the amine salt of the uronic acid-containing polysaccharide with sulfuric acid. In a preferred embodiment, the sulfuric acid is present in about 1 to about 25 weight excess over the uronic acid-containing polysaccharide and, more preferably, in about a 3 to about a 15 weight excess to the uronic acid-containing polysaccharide. Thereafter, the first solution is contacted with an N,Nxe2x80x2-carbodiimide to form a second solution. Examples of suitable N,Nxe2x80x2-carbodiimides include, but are not limited to, the following: dicyclohexylcarbodiimide (DCC); diisopropylcarbodiimide (DIC); 3-ethyl-1(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 1-cyclohexyl-3-(2-morpholoethyl)carbodiimide p-toluene sulfonate (CMC). Typically, the N,Nxe2x80x2-carbodiimide is present in an amount that is equimolar to the amount of sulfuric acid used. The N,Nxe2x80x2-carbodiimide is preferably added in three equal portions at equally spaced intervals, such as 20 minute intervals. In a preferred embodiment, DCC or EDC is the N,Nxe2x80x2-carbodiimide used.
The second step of the above process is preferably carried out in a dipolar, aprotic solvent. Suitable dipolar, aprotic solvents include, but are not limited to, the following: dimethylformamide (DMF), dimethylsulfoxide and pyridine. In a presently preferred embodiment, dimethylformamide is employed as the solvent. In addition, the second step is preferably carried out at a temperature ranging from about 10xc2x0 C. to about 35xc2x0 C. and, more preferably, at a temperature of about 25xc2x0 C.
After the addition of the N,Nxe2x80x2-carbodiimide, the second solution is allowed to stand for a period of about 1.0 to 3.0 hours and, more preferably, for a period of about 1.5 hours. Thereafter, any precipitate formed is removed by filtration. One to three volumes of acetone or dichloromethane and, more preferably two volumes of acetone are added to the filtrate or, in the case where no precipitate is formed, to the second solution in order to form a precipitate. The pH of the second solution is adjusted to a pH of about 12 and, more preferably, to a pH above 13. This is typically carried out by adding about 0.1 to 3.0 N NaOH and, more preferably, about 0.5 to 2.0 N NaOH to the second solution until the pH is above 12 and, more preferably, above 13. The second solution is allowed to stand for a period of about 0.5 to 1.0 hour at a temperature of about 20xc2x0 C. to 30xc2x0 C. Thereafter, the pH of the second solution is lowered to a pH of about 6.0 to about 8.0 and, more preferably, to a pH of about 7.0.
In the third step of the above process, applicable primary to heparinoids, the O-sulfated uronic acid-containing polysaccharide is further N-sulfated by contacting the second solution with a sulfating agent. In a presently preferred embodiment, the N-sulfation is carried out according to the method of Lloyd, A. G., et al., Biochem. Pharmacol., 20:637-648, the teachings of which are incorporated herein by reference. As such, in the third step of the above method, the sulfating agent is a complex including, but not limited to, sulfur-trioxide with an organic base and chlorosulfonic acid in pyridine. In a presently preferred embodiment, the complex is pyridine sulfur-trioxide or trimethylamine sulfur-trioxide. In a presently preferred embodiment, trimethylamine sulfur-trioxide is the sulfating agent employed. Typically, the second solution is mixed with water to form a 1% to about 3% and, more preferably, 2% O-sulfated uronic acid-containing polysaccharide aqueous solution. To this 1% to 3% O-sulfated uronic acid-containing polysaccharide aqueous solution is added sodium bicarbonate (NaHCO3) to make about a 0.25-0.5 M NaHCO3 solution. The N-sulfation of the O-sulfated uronic acid-containing polysaccharide is performed at a temperature of about 50xc2x0 C. to about 60xc2x0 C. for a period of about 3 to 6 hours by contacting the second solution with the sulfating agent such that the ratio of the sulfating agent to that of the uronic acid-containing polysaccharide is about 1:0.5 to about 1:2.
Those of skill in the art will readily appreciate that the resulting suronic acid-containing polysaccharide compounds can be subjected to further purification procedures. Such procedures include, but are not limited to, gel permeation chromatography, ultrafiltration, hydrophobic interaction chromatography, affinity chromatography, ion exchange chromatography, etc. Moreover, the molecular weight characteristics of the suronic acid-containing polysaccharide compounds of the present invention can be determined using standard techniques known to and used by those of skill in the art as described above. In a preferred embodiment, the molecular weight characteristics of the suronic acid-containing polysaccharide compounds of the present invention are determined by high performance size exclusion chromatography in conjunction with multiangle laser light scattering (HPSEC-MALLS).